Abstract Developing the required biomarkers that define IL-23R target engagement and effect on downstream signaling for late-stage drug development and early clinical proof-of-concept. Inflammatory bowel disease (IBD) affects about 0.5% of the world's population. Due to its early onset and lack of an adequate cure, this disease requires lifelong treatment. IBD affects the gastrointestinal (GI) tract and manifests as two subtypes: Crohn's disease (CD) and ulcerative colitis (UC). In recent years, anti-TNF biological agents have transformed the treatment of IBD, but these are not ideal drugs, requiring administration by injection, and in some instances hospitalization for intravenous infusion. These agents have numerous side effects, including increased infection rates; additionally, anywhere from 10-30% of patients either lose response (through the production of neutralizing antibodies) or become intolerant (e.g., site reactions). Long half-lives of injected antibodies, which can result in TNF blockade over sustained periods, can exacerbate these issues and make it hard to control drug exposure to minimize safety issues. Although current anti-TNF drugs possess similar modes of action, switching from one agent to another is an established treatment approach for patients who become unresponsive or intolerant. Stelara (ustekinumab) targets both the IL-12 and IL-23 pathways and is efficacious in Phase III Crohn's disease clinical trials in TNF-resistant patients. However, there is some concern about cardiovascular safety events, as illustrated by the removal from the market of briakinumab (which also targets IL-12 and IL-23) due, in part, to increased cardiovascular risk. IL-23 is produced locally in the intestine and plays a fundamental role controlling intestinal mucosal inflammation. Hence, selectively modulating the IL-23 pathway locally in diseased tissue is the preferred strategy. Such an approach would provide high concentrations of drug in diseased tissue and block IL-23 function locally in the intestine. During the Phase I SBIR , we developed a potent (2 nM), orally stable antagonist (PN1140) of the IL-23 receptor (IL-23R) that is efficacious when orally delivered in a TNBS-induced model of colitis. PN1140 prevented body weight loss, reduced the colon weight-to- length ratio, and (most importantly) improved colon macroscopic pathology. PN1140 was predominantly restricted to GI tissue with minimal systemic exposure. The overall objective of this Phase II SBIR proposal is to develop methods for characterizing in vivo target engagement, including pharmacokinetic and pharmacodynamic methods to characterize the binding of PN1140 to IL-23R in various compartments and how binding affects downstream biomarkers and efficacy. These biomarkers will be used to aid compound and dose selections, and to provide early-stage clinical proof-of-concept. The specific objectives are to: 1) Develop the required target engagement methods to enable quantification of binding of PN1140 to IL-23R-bearing cells; 2) Identify the required pharmacodynamics biomarkers that would reflect the downstream biological changes upon target engagement; and 3) Correlate target engagement and pharmacodynamics biomarkers with efficacy readouts in TNBS models of colitis in rats. This Phase 2 SBIR program is supported by a team that has a track record in the oral delivery of constrained peptides, a proven capacity in translating early-stage research to clinical outcomes, a group of scientific and clinical advisors with significant experience in IBD, and the appropriate research environment. In this Phase II SBIR proposal, we describe the development of the appropriate biomarkers that will establish early proof-of-concept and the effective human dose range through an assessment of target engagement and pharmacologic activity in early-stage human trials. These are important steps towards our ultimate objective of demonstrating clinical benefit in late-stage clinical trials. These biomarkers will permit the assessment of mechanism-specific and disease-related parameters in blood, fecal, and/or colon biopsy samples.